1) Background The Golgi complex is a subcellular organelle that is essential to every cell, from yeast to complex mammalian cells. All membrane and secreted proteins transit through the Golgi complex where they undergo chemical modifications such as glycosylation. The Golgi complex is also the site where these proteins are sorted and targeted to their destination, a particularly important function in large cells such as muscle, which contains distinct membrane domains. The multiplicity of functions of this organelle is linked to its peculiar structural organization which in muscle changes dramatically during differentiation and regeneration. Very little is known of the mechanism of these changes. The organization of the Golgi complex of muscle also depends on the pattern of contractile activity and this regulation is not understood at all. Our goal is to understand how the distribution of the Golgi complex in muscle is regulated and is linked to the functional needs of muscle. In the past we have uncoverered basic aspects of the changes that take place during differentiation. We have established that the Golgi complex of each myoblast fragments into hundreds of smaller independent Golgi complexes placed around the nuclei and throughout the cytoplasm of the multinucleated myotubes and muscle fibers. These small Golgi elements are not distributed randomly but are retained next to endoplasmic reticulum sites specialized in the export of proteins to the Golgi complex. This specific localization suggested that the fragmentation of the Golgi complex during muscle differentiation resembles the fragmentation that the Golgi complex undergoes when microtubules are depolymerized. We have indeed demonstrated the similarity between these processes, therefore identifying changes in the microtubule cytoskeleton as a key factor in the changes that take place during differentiation. Patterned activity is an important regulator of muscle metabolism and contraction but it had never been linked to the organization of the protein secretory pathway. We have discovered that the distribution of Golgi complex, endoplasmic reticulum exit sites and microtubules is plastic in mature muscle fibers and responds to the pattern of contractile activity, causing a fiber type-dependent organization. We hypothesize that this plasticity is important and allows muscle to fulfill different metabolic demands depending on patterned activity. 2) Objective of present studies Our past work provided, for the first time, a description of the organization of the Golgi complex in muscle fibers. In order to understand how it is controlled by external factors such as patterned activity, we need to learn more about the interaction between cytoskeletal elements and Golgi complex organization. We also need to determine how their interaction is regulated at the molecular level and uncover the signaling pathways that are involved. 3) Results during the past year We have continued to investigate the changes in the organization of microtubules during muscle differentiation. We have focused on the protein ninein which, in endothelial cells, accompanies microtubules during their redistribution following cell polarization. We have been able to detect ninein in both undifferentiated and differentiated muscle and we have shown that it becomes redistributed during muscle differentiation. However, ninein does not seem to accompany small microtubules released from the centrosome shortly after their synthesis. This suggests that the mechanism of microtubule reorganization in muscle is distinct from that observed in endothelial cells and that ninein may play a different role in muscle. We have also started to explore signaling pathways that may regulate the Golgi complex organization. We have observed that lithium chloride affects the reorganization of both centrosomal proteins and Golgi complex during muscle differentiation. Lithium chloride is an inhibitor of GSK3-beta, an important kinase. It also affects stable microtubules. We are now using pharmacological tools and cDNA constructs to identify effectors of this pathway, which has been studied in epithelial cells migrating into a wound. Interestingly, we have observed that lithium chloride, previously reported to block muscle differentiation, blocks fusion of myoblasts but not their molecular differention as evidenced by normal induction of the muscle protein myogenin. 4) Conclusions and significance The Golgi complex is an essential organelle which shows great plasticity in skeletal muscle in response to changing physiological conditions and in pathological situations. It is therefore important to understand how this organelle is regulated. But because it has mostly been studied in proliferating, non-differentiated cells, we know little of the changes it undergoes in complex differentiated tissues. We have identified tools, such as lithium chloride and other effectors of the GSK3-beta pathway, which affect the organization of the Golgi complex during muscle differentiation and will allow us to start investigating this problem at the molecular level.